Volume 3, Issue 11, November – 2018 International Journal of Innovative Science and Research Technology

ISSN No:-2456-2165

Biodegradation of Toxic Effluent Using Immobilized

Beads of Rhizobium
Jenisha J J1Seshan D and Sheela S S1*
Author’s affiliation
1. Department of Biotechnology, Loyola College, Nungampakkam, Chennai - 600034
Corresponding author
Dr S. Sheela
Coordinator, Department of Plant Biology and Biotechnology
Loyola College, Chennai – 600034

Abstract:- The effluent industries is a major source of
water pollution. So their treatment before discharge is
very crucial and biodegradation where microorganisms
break down the azo bonds to form its nontoxic basic
element is the most effective way. Toxic effluent present in
the industrial effluent has highly carcinogenic substances
like aromatic amines. The study involved isolation of
Rhizobium, molecular and biochemical characterization,
immobilization of Rhizobium in sodium alginate capsules
and the examination of biodegradation of toxic chemicals
present in the industrial effluent using the encapsulated
Rhizobium, decolourization of methylene dye using
immobilized beads. The spectrophotometric assessment of
dye degradation was by measuring at optical density
530nm. Biodegradation of toxic effluent by immobilized
Rhizobium is the novel technology that may be applied for
the treatment of waste water containing a mixture of
different dyes.
Keywords:- Rhizobium; Immobilization; Biodegradation;
Decolourization.
I. INTRODUCTION
Water pollution is major problem in the global context
and has even been suggested to be the leading cause of death
and disease worldwide, especially in developing countries.
High production and use of dyes generate colored waste
waters that pollute rivers: their presence in surface water
blocks solar radiation from reaching aquatic organisms, thus
affecting negatively tea balance of ecosystems [1]. The dyes
like azo dye are toxic are highly carcinogenic. These toxic
dyes are unfortunately derived to be resistant to many
degradation process, and those are highly toxic and may lead
to cancer [2].
Rhizobium is a well-known bacteria that act as the
primary symbiotic fixer of nitrogen. These bacteria infect the
roots of leguminous plants leading to the formation of lumps
or nodules where the nitrogen fixation takes place. Set of
genes in the bacteria control different aspects of the nodulation
process. One Rhizobium strain can infect certain species of
legumes but not others. When legume plants encounter low
nitrogen conditions and want to form a symbiotic relationship
with Rhizobia they release flavonoids into the soil. Rhizobia
responds by releasing nodulation factors which stimulates the
formation of nodules in plant roots which in turn helps in
symbiotic relationship.Various physico chemical methods are
used to detoxify toxic effluent to achieve degradation. These
include filtration, coagulation, carbon activated and chemical
flocculation. These methods are effective but they are
expensive and involve the formation of a concentrated sludge
that creates a secondary disposal problem. Microbial
degradation is an environmentally friendly and a cost
competitive effective to chemical decomposition process [3].
Annually more than 50% of azo dyes are used [9].
Around 2000 of them are used in textile industry, leather,
plastics, paper, cosmetics and food industries [7]. Azo dyes
are characterized by the presence of one or more azo groups
substituted with aromatic amines [7]. A substituent usually
found in the azo dyes is the sulfonic acid. The azo dyes has
this substituent are commonly known as sulfonated azo dyes.
These azo dyes are used in various industries [8]. The fixation
rate of these reactive dyes in dying is as low as 50%, which
results the release of 10-15% water soluble azo dyes into the
environment through waste water discharge. Sulfonated and
unsulfonated azo dyes has negative bad effect on waste water
and some of these compounds and biodegraded substituents
are toxic, carcinogenic, and mutagenic. These dyes have more
number of structural diversity and they are made to resist
physical, chemical and microbial attack [4].
II. MATERIALS AND METHODS
 Media preparation
Yeast Extract Mannitol Agar (YEMA/Congo red) is a
selective media commonly used for the cultivation of soil
microorganisms like Rhizobium species and studying root
nodulation. It contains Mannitol, yeast extract, Dipotassium
phosphate, magnesium sulphate, sodium chloride, agar and
congo red. Congo red acts as an indicator.
 Isolation of bacterial strain
Root nodules of Arachis hypogea were collected from
Loyola College, Chennai and Tamil Nadu. The collected root
nodules were washed with 95% alcohol for 30 secondsto
remove other microorganisms and again washed with sterile

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Volume 3, Issue 11, November – 2018
water. Using sterile forceps the root nodules were transferred
into sterile mortar and pestle and gently it was grounded. The
root nodule suspension was diluted by adding sterile water and
serially diluted. A loopful of culture from 10-3 dilution was
taken and spread plate technique was performed on Yeast
Extract Mannitol Agar plate. The inoculated petri plates were
incubated for 24 hours at 37ºC. After 24 hours the plates were
observed.
 Biochemical and molecular characterization
Series of biochemical tests were done which includes
Gram’s staining, Oxidase test, Catalase test, methyl red,
Voges-Proskeur, Urease test, nitrate reduction test, citrate
utilization test, Indole acetic acid test and triple sugar iron test.
The isolated Rhizobium was inoculated in 100 ml freshly
prepared Yeast Extract Mannitol Broth. The inoculated broth
was incubated at 37ºc for 24hrs. The bacterial genomic DNA
was isolated using Hyper Genomic DNA isolation kit method.
Complete reagent recipe for polymerase chain reaction
includes PCR grade water 19µl, 1.5µl forward and reverse
primer, template DNA 3µl and PCR master mix 25µl. All the
components of reaction mixture were mixed well except the
sample DNA solution. The mixture was loaded into PCR tubes
27forward primer and 1490 Reverse primer.
 Immobilization of Rhizobium
The isolated Rhizobial species was immobilized in 3%
sodium alginate.
 Biodegradation of toxic effluent
The effluent along with the media was incubated with
immobilized beads of Rhizobium for about 48 hrs at 37° C.
 Decolorization
The immobilized bacterial culture was inoculated in the
conical flask containing 100 mL yeast mannitol broth and was
grown in static or shaking conditions(120 rpm) and incubated
at 30°c for 48 hrs. The optical density of the dye was taken
before and after incubation by using UV- Visible
Spectrophotometer.
III. RESULTS
The Rhizobial cells were isolated from the root nodules
of Arachis hypogea(Fig 1: A). The growth of Rhizobium was
clearly visible in Yeast Mannitol Agar containing Congo red.
The Rhizobial colonies were observed as mucoid, translucent
and slightly pink colonies(Fig1: B). Gram staining showed
Gram negative rods (Fig1: C). The isolated microorganism
was identified as Rhizobium by the biochemical characters
from Bergey’s Manual (Fig 1: D to H and Table 1). The
isolated genomic DNA was electrophoresed on 0.8 % Agarose
gel which was stained with Ethidium Bromide. After running
the gel, it was observed under UV transilluminator. Clear
bands were observed.The PCR reaction product was analyzed
by agarose gel electrophoresis and the DNA of the expected
size was purified and sequenced. An amplicon of 700 bp was
obtained on PCR amplification of the DNA with specific
forward and reverse primers (Fig 2).The presence and amount
IJISRT18NV308
International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
of carbon containing chemicals were assessed by GC-MS (Fig
3). The mass of certain chemicals shows high. The mass of
Benzamide,4-butyl-N-(2-ethylphenyl) was the highest of
388.30 m/z and the mass of 5,6-Dihydro-5-methyluracil was
the least of 42.20 m/z. Totally 15 different chemicals were
identified in the sample (Table 2).Rhizobium was immobilized
in 3% sodium alginate. The capsules appeared spherical in
shape and uniform diameter.After treating the effluent with the
immobilized isolate GC-MS analysis was done (Fig 5). The
number of chemicals had reduced from 15 to 2 (Table 3).
Ocatanoic acid and hepatanoic acid-oxo-trimethylsilyl ester
were the two chemicals found in the sample after
degradation.The intention of this study was to check the
ability of organism for decolourization of dye. The
immobilized isolate decolourized the dye in the sample
IV. DISCUSSION
Rhizobia or bacteria isolated from the root nodules are
rod shaped and are Gram negative. Rhizobia are Gram
negative, and are mobile. Uneven Gram staining is frequently
encountered with Rhizobia, depending on the age of the
culture. Cells from a young culture and nodule bacteriods
usually show even Gram staining while older and longer cells
give a banded appearance with unstained areas. Most Rhizobia
only weakly absorb Congo red (diphenyldiazo-bis-α-
napthylamine sulfonate) dye, which is included in culture
media for isolating Rhizobia. Optimum temperature for the
growth of Rhizobia is 25-30ºC. Rhizobium are bacteria
capable of forming a nitrogen-fixing symbiosis with
leguminous plants [5].
The Rhizobial isolate was identified on the basis of their
morphological, cultural, microscopic and biochemical
characteristics including size, shape form of the bacterial cells,
presence or absence of spores, Gram reaction, Urease, Nitrate
reduction, etc. the standard description given in “ Bergey’s
Manual of Determinative Bacteriology” [6]. The molecular
characterization was done after Polymerase Chain Reaction.
The e-value was found to be 0.0 and the similarity with
bacillus species was 96%. Thus it has no 100 % similarity
with any other organism. The biochemical test identifies this
organism as Rhizbium hence it should be an unidentified
Rhizobium species.
The effluent had 15 different types of chemicals. Which
includes allyl methyl sulphide, Benzenedaiazepin,
octadecamethyl, octaatomic sulphur. These are toxic
chemicals which causes various health issues. Contact with
these chemicals can cause skin irritation. Inhalation of these
chemicals will cause health hazards. After contact with the
skin, wash immediately with plenty of water. Very hazardous
in case of ingestion, hazardous in case of skin contact, of eye
contact, of inhalation. These are may be combustible at high
temperature. After the degradation the characteristics of the
sample had changed. After degradation only 2 chemicals were
found, octanoic acid mass was 117.10 m/z and hepatanoic
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Volume 3, Issue 11, November – 2018 International Journal of Innovative Science and Research Technology
acid-oxo-trimthylsyl ester, masss was 75 m/z (Kabra et al.
2012).
The entrapment technology, immobilization had helped
the Rhizobium to grow and multiply within the matrix. Thus
the immobilized beads of Rhizobium were able to degrade the
toxic chemicals found in the sample. The main advantage of
the immobilization of Rhizobium using sodium alginate is that
the beads can be re-used for the treatment of the effluent [11].
Thus various types of chemicals discharged from various
industries can be treated by Rhizobium. The effluent treated
with the immobilized Rhizobium will not cause any
environmental hazard. Hence this type of eco friendly
treatment is necessary for the healthy environment. This type
of biological method can be used for any kind of effluent
treatment.
Rhizobial beads are also used for the reduction of dye
(1% methylene blue) [10]. Both shaking and static conditions
showed dye degradation. In one set of degradation eight beads
were used. In another one set of degradation 12 beads were
used. Degradation of dye in both static and shaking condition
were observed. Degradation in the shaking condition was
faster when compared to the shaking condition. Degradation in
the shaking condition with 12 beads showed faster than the
degradation in the shaking condition with 8 beads. Thus this
may be due to number of Rhizobial culture was higher in 12
beads than the 8 beads. Thus the degradation efficiency can be
increased by adding 2 ml of culture. It took 48 hours to
degrade 93% of methylene blue dye under shaking condition
with 12 beads. By increasing the number beads the time
required for the degradation can be reduced. May be the dye
was absorbed by sodium alginate which was used to entrap the
Rhizobium. Control without sodium alginate fails to explain
this. Sodium alginate is a gum derived from seaweed.
INDOLE CITRATE METHYL VOGES
ISOLATE
TEST UTILIZATION RED PROSKEUR
R1 - + - -
Table 1:- Biochemical Tests
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ISSN No:-2456-2165
Methylene blue is a dye used in laboratories. High
production and use of dyes generate coloured waste waters
that pollute rivers; their presence in surface water blocks solar
radiation from reaching aquatic organisms, thus affecting the
balance of aquatic ecosystems. In addition, the release of dyes
into water streams may result in formation of toxic and
carcinogenic degradation products. Hence, the removal of
dyes from industrial effluent is an important and necessary
process [12]. Discoloration of water has become one of the
major issues in waste water treatment as many industries use
dyes to colour products such as textiles, rubber, paper,
plastics, leather, cosmetics, food and minerals[13].
V. CONCLUSION
In the present study, the isolated microbe degraded toxic
chemicals present in the effluent and also decolorized
methylene blue. Thus this micro organism can be used to treat
the effluent from the various industries. The microbe was
immobilized within the sodium alginate. Thus the organisms
will be trapped inside the matrix. Thus the beads can be re-
used to treat the effluent. Direct culture of the isolated microbe
can also be added to treat the effluent. This method of
treatment is eco-friendly. This method of treatment is eco-
friendly. Biological treatment method is highly efficient also.
These microbes need suitable substrate to grow and multiply.
Various carbon and nitrogen sources can be provided for the
growth of the organism. Those organisms will take time to
multiply and thus degradation time is also longer when
compared to other methods. But the microbial biodegradation
is much safer than the other methods of degradation.
UREASE NITRATE OXIDASE CATALSE
TEST REDUCTION TEST TEST
+ + + +
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ISSN No:-2456-2165

RT Area Mass Super

S. No Compound
(min) (%) m/z Impossibility

1 Sulphide, allyl methyl 7.415 3.47 73.20 53

2 5,6-Dihydro-5-methyluracil 8.745 8.75 42.20 72

3 2-Propylnoanoic acid 10.058 7.17 73.10 50

4 Piperidin-4ol,2,3,dimethyl trans 12.572 10.52 114.10 38

5 Cyclic octaatomic sulphur 13.738 2.05 63.90 56

6 Imidazole 20.108 4.82 42.20 78

7 1,4-Benzenedaiazepin-2-one 26.314 2.18 73.20 38

8 Cyclolnonsailoxane, octadecamethyl 28.168 2.85 207.0 43

9 Hexa (methoxymethyl) melamine 30.274 3.27 207.10 38

Benzeneaceticacide, alpha, 3, 34, tris
10 30.788 2.00 207 38
Trimethysilyl ester

11 18,19-secolupan-3-ol,3beta 32.82 2.54 281.00 14

12 Benzamide,4-butyl-N-(2-ethylphenyl) 35.86 3.25 388.30 27

13 Cyclodecasiloxane, eicosamethyl 37.507 2.46 207.20 25

14 Bianthrone 38.021 2.19 384.50 43

15 Octasiloxane-hexadecamethyl 38.219 1.94 207.20 38

Table 2:- Characteristics of the Effluent Before

RT Area Mass
S.NO Compound Super Impossibility
(min) (%) m/z

1 Octanoic acid 17.180 61.96 117.10 30

2 Hepatanoic acid-oxo-trimethylsilyl ester 17.500 38.04 75.10 25

Table 3:- Characteristics of the Effluent after Biodegradation

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Volume 3, Issue 11, November – 2018 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165

Fig 1:- A) Root nodules collected from Arachis hypogea;B) isolated colonies of Rhizobium C) Gram stainingD – H) Biochemical
Tests such as, D) urease test, E) nitrate reduction, F) citrate utilization, G) catalase test and H) oxidase test.

Fig 2:- PCR amplification of Genomic DNA from isolates - Lane L – 100 bp marker; Lane 1 – R1

Fig 3:- Spectra indicating the characteristics of the effluent sample

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Volume 3, Issue 11, November – 2018 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165

Fig 4:- A) Decolourization of 1 % methylene blue in shaking condition within 48 hours (8beads); B) Decolourization of methylene
blue within 48 hours in static condition (8 beads); C) Decolourization of 1% methylene blue within 48 hours under shaking condition
(12 beads); D) Decolourization of 1% methylene blue within 48 hours under static condition (12 beads) 10-13.

Fig 5:- Characteristics of the effluent after biodegradation (GC-MS analysi

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Volume 3, Issue 11, November – 2018 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
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